Hypoxia figures importantly in the biggest of health issues. It is responsible for the damage caused by the ischemia accompanying cardiac infarct and stroke, and it plays a central role in limiting tumor growth as well as blunting the actions of important chemotherapies. While we have a mechanistic understanding of hypoxic regulation of transcription by Hypoxia Inducible Factor (HIF), other modes of response to oxygen deprivation are still poorly understood. How does the mitochondrion, the major consumer of oxygen in the cell, adjust to shortfalls in oxygen supply, and how does it signal to the rest of the cell, demanding accommodations to these shortfalls? Reactive oxygen species produced by mitochondria have been ascribed the responsibility of communicating oxygen stress to the cell, but precise pathways of signal generation, transduction and action have not been established. Recognizing the complexity of the issues and the fundamental nature of the questions, it seems that the powerful genetics available in model organisms could make a major contribution. We found that nitric oxide (NO) mediates immediate responses to hypoxia in Drosophila, including an arrest of embryos in a reversible state of suspended animation. We have devoted ourselves to the development of a powerful and greatly simplified experimental context in which we can dissect the NO-signaling mechanisms. Our finding that NO regulates innate immune responses in Drosophila revealed a hypoxia-immunity connection, and gave us the simplification we sought. Immune reporters signal the action of NO, and the response to hypoxia can be recapitulated in cultured cells amenable to powerful RNAi screens. In this proposal, we will combine in vivo studies and analysis of cell culture models to investigate NO-mediated signaling. In vivo studies will probe how infection induces NOS, and how NO signaling contributes to the immune response. In Drosophila S2 cells, we will examine the basis of hypoxia-induced NO signaling that appears to originate in the mitochondria, and will trace the transformation and transport of this signal as it is conveyed to the cytoplasm. Finally, we will define the genes and pathways by which mitochondria sense hypoxia and signal the dramatic changes within the mitochondria, and will the coupling between mitochondrial changes and the signals emanating from the mitochondria that have such huge impacts on survival of cells and the well-being of man. These studies will give us a mechanistic understanding of a major component of the hypoxia response, contribute to the understanding of the biological diversity in tolerance to hypoxia, and give us approaches to manipulate hypoxia sensitivity to potentially benefit issues of important health concern. PUBLIC HEALTH RELEVANCE The most common life-threatening health problems, cardiac infarct and stroke, cause damage by interrupting oxygen supply, yet we understand little about the biological responses to the resulting acute hypoxia. We will dissect the mechanisms of response to hypoxia in a powerful model genetic system, Drosophila. Our findings have the potential to influence treatment of cardiac infarct and stroke, and could have an impact on areas as diverse as sustaining organs for transplantation, and cancer therapies.